IHC background

sean troth sean.troth at colostate.edu
Thu Feb 28 14:37:09 EST 2002

Have you looked at a section with no antibody to see if there is simply 
an autofluorescence problem?  Autofluorescence of red cells and collagen 
is very common.  Depending on the filter/wavelength you're using you can 
quench some autoflourescence by using a chromagen counterstain such as 
trypan blue.  Another way to determine autofluorescence is to switch 
between filters (i.e. FITC to TRITC).  Cells will continue to 
autofluoresce between filters whereas true chromagen fluorescence will 
only fluoresce in the appropriate filter.  The exception to this being 
that you can get some "burn through" to other filters by using extremely 
large amounts of fluorochrome (i.e.via tyramide over-amplification).

Lastly you're already using a maximal conc. of blocking agent.  Its my 
opinion that you cannot block your way out of nonspecific background 
problems.  You should begin by diluting the primary antibody perhaps in 
conjunction with using a signal amplification system (i.e. tyramide).

sean troth

Dr. Douglas Darling wrote:

>I am using an FITC-conjugated secondary antibody on histological
>sections of mouse embryos.  We get great labeling with our primary
>antiserum.  However, all the blood cells also label.  The blood cells
>fluoresce brightly even when no primary antibody is used.  I use 10%
>serum-0.1% triton to block.  There is obviously some non-specific
>binding of the secondary Ab-FITC to the blood cells.  I tried 4
>different secondary Ab conjugated to either FITC or TRITC.  HOW CAN I

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